Method and apparatus for separating cells by sedimentation velocity

Abstract

A method and apparatus for separating cells according to differences in sedimentation velocity comprises a vessel preferably constructed from a cylindrical body of ring formation, the height of which is substantially less than its diameter, and from top and bottom plates sealingly and removably attached to the ring. The vessel is preferably mounted on a tiltable platform for controlled movement between tilted and horizontal positions and has a small horizontal cross-sectional area in the tilted position relative to such area in the horizontal position. The vessel is provided with two ports; a lower port communicating with a lowermost level of the vessel while in the tilted position and an upper port communicating with the uppermost level in the tilted position. Flow distributing structure is provided preferably between each port and the interior of the vessel whereby the force of liquid flowing into the vessel through a port is dissipated. A gradient liquid and a cell suspension are loaded into the vessel while it is in a tilted position. Thereafter, the vessel is rotated to the horizontal position for cell separation. After separation, the vessel is reoriented to the tilted position, and the gradient liquid containing separated cells is collected as various fractions.

I claim: 
     
       1. Apparatus for separating cells of differing sedimentation velocities, comprising a closed separation vessel having an open and unobstructed interior and adapted to be mounted for tilting from and back to horizontal position; a relatively very small lower port located at the lowermost level of the interior of said vessel when said vessel is in its tilted position; a similar upper port located at the uppermost level of the interior of said vessel when said vessel is in its tilted position, the opening of at least the lower port into the interior of the vessel being offset from the interior of the vessel; and flow distributing means having flow distributing structure located between the lower port and the interior of the vessel and offset from the interior of the vessel for fanning out and decreasing the velocity of the incoming flow and thereby reducing the tendency of incoming liquid to disturb isodense layers of liquid contained in the vessel. 
     
     
       2. Apparatus according to claim 1, wherein the flow distributing structure comprises a baffle against which the flow from the port is directed for expansion and velocity reduction before it enters the interior of the vessel. 
     
     
       3. Apparatus according to claim 1, wherein the flow distributing structure comprises a shallow expansion chamber leading from the port into the interior of the vessel for fanning out and decreasing the velocity of the incoming flow before and as it enters the interior of the vessel. 
     
     
       4. Apparatus according to claim 3, wherein the flow distributing structure also comprises a baffle against which the flow from the port is directed for diverting the flow into and through the expansion chamber for entry into the interior of the vessel. 
     
     
       5. Apparatus according to claim 4, wherein the separation vessel comprises a cylindrical body of ring formation and flat top and bottom walls closing said body, the ports passing through said top and bottom walls, respectively, at diametrically opposite locations which are uppermost and lowermost, respectively, when the vessel is in tilted position, at least the lower port opening into the corresponding end face of said body, which serves as the baffle, and the expansion chamber of the flow distributing structure being formed in the inside face of the said bottom wall at its juncture with the cylindrical body. 
     
     
       6. Apparatus according to claim 5, wherein the lower port is directed against the baffle at an angle of substantially ninety degrees. 
     
     
       7. Apparatus according to claim 6, wherein the expansion chamber is circular and has about one-half of its area disposed in the junction between the cylindrical body and the bottom wall. 
     
     
       8. Apparatus according to claim 1, wherein the separation vessel comprises a cylindrical body of ring formation and flat top and bottom walls closing said body, the ports passing through said top and bottom walls, respectively, at diametrically opposite locations which are uppermost and lowermost, respectively, when the vessel is in tilted position. 
     
     
       9. Apparatus according to claim 8, additionally comprising mounting means for the separation vessel, said mounting means comprising a platform adapted to receive the separation vessel in horizontal position, and means for moving said platform between horizontal and tilted positions and vice versa, said moving means being adapted to accelerate movement of the platform to about the midpoint of said movement and to decelerate said movement beyond said midpoint. 
     
     
       10. Apparatus according to claim 9, wherein the moving means comprises one-half sinusoidal electric motor means arranged to move the platform to the tilted position and back to the horizontal. 
     
     
       11. Apparatus according to claim 8, wherein the top and bottom walls are removably attached to the cylindrical body; and wherein sealing means are interposed between said body and said walls. 
     
     
       12. Apparatus according to claim 11, wherein the sealing means are respective O-rings set into receiving grooves formed around the outer margin of the end faces, respectively, of the cylindrical body. 
     
     
       13. Apparatus for separating cells of differing sedimentation velocities, comprising a closed separation vessel having an open and unobstructed interior; a relatively very small lower port located at the lowermost level of the interior of said vessel, the opening into the interior of the vessel of at least the lower port being offset from the interior of said vessel; and flow distributing means associated with at least said lower port and having flow distributing structure located between the port and the interior of the vessel and offset from the interior of the vessel for fanning out and decreasing the velocity of the incoming flow, thereby reducing the tendency of the incoming liquid to disturb isodense layers of liquid contained in the vessel. 
     
     
       14. Apparatus according to claim 13, wherein the flow distributing means also comprises flow distributing structure located between the upper port and the interior of the vessel. 
     
     
       15. Apparatus according to claim 1, wherein the flow distributing means means comprises flow distributing structure located between the upper port and the interior of the vessel. 
     
     
       16. A method for separating cells of differing sedimentation velocities, comprising the steps of tilting from horizontal position a separation vessel constructed in accordance with claim 1; introducing a density gradient liquid through the lower port and distributing its flow by means of a baffle or expansion chamber or both before and as it enters the interior of the vessel; filling the vessel with said gradient liquid until flow out of the upper port begins; loading the vessel with a cell suspension containing cells which are to be separated according to differences in sedimentation velocity, said loading being by way of the flow distributing means so said cell suspension will be distributed before and as it enters the interior of the vessel; moving the vessel back to horizontal position so that the isodense layers of the gradient liquid decrease in thickness with respect to their thickness when the vessel is in the tilted position; leaving the vessel in the horizontal position for a sufficient period of time to allow the cells in the cell suspension to separate on the gradient according to their respective sedimentation velocities; again tilting the separation vessel so as to increase the thickness of the isodense layers; and collecting the isodense layers of gradient liquid as various fractions, by removing the gradient liquid through the lower port.